Abstract: Objective To investigate the effect of crocin on bupivacaine-induced neurotoxicity and the role of the PI3K/Akt signaling pathway. Methods Human neuroblastoma SH-SY5Y cells were normally cultured and randomly divided into the following groups: control (Con), crocin (Cro, 200 μmol/L), bupivacaine (Bup, 1 mmol/L), and Bup+Cro groups. MTT assay was used to assess cell viability, Ki67 immunofluorescence was employed to detect cell proliferation, and flow cytometry was used to analyze cell apoptosis. The activities of Caspase-3, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) content was measured by corresponding kits. ROS production was detected by DCFH-DA assay. Western blot was used to detect the expression of apoptosis-related proteins and PI3K/Akt pathway proteins. Results Bupivacaine (0, 0.5, 1.0, and 1.5 mmol/L) inhibited cell viability in a dose-dependent manner (P<0.05), while crocin alone had no significant effect on cell viability (P>0.05). After Bup+Cro co-treatment, cell viability was gradually increased as the crocin concentration (50, 100, and 200 μmol/L) increased. Compared with the Cro group, the Bup group showed significantly reduced cell proliferation and increased apoptosis rate, with elevated Caspase-3 activity, Bax protein expression, MDA content, and ROS production, while Bcl-2 expression, GSH-Px, SOD, and CAT activities, and the p-Akt/Akt and p-GSK-3β/GSK-3β ratios were significantly decreased (P<0.01). Compared with the Bup group, the Bup+Cro group showed increased cell proliferation, and decreased apoptosis rate, with reduced Caspase-3 activity, Bax protein expression, MDA content, and ROS production, while Bcl-2 expression, GSH-Px, SOD, and CAT activities, and p-Akt/Akt and p-GSK-3β/GSK-3β ratios were increased (P<0.05). After pretreatment with Akt inhibitor tricirbine (API), the Bup+Cro+API group showed decreased cell viability, and increased cell apoptosis rate, with elevated Caspase-3 and Bax expression, and increased ROS production, while Bcl-2 expression was reduced (P<0.01). Conclusions Crocin may alleviate bupivacaine-induced neurotoxicity by inhibiting the activation of the PI3K/Akt pathway, promoting SH-SY5Y cell proliferation and inhibiting cell apoptosis and oxidative stress.